Streptococcus agalactiae, also known as Group B Streptococcus ("GBS") has long been associated with mastitis in dairy animals. More recently, GBS has been recognized as a serious pathogenic agent in humans; GBS is a causative agent in meningitis, bacteremia, endocarditis, bronchopneumonia, arthritis, peritonitis, wound infections, and urinary tract infections in adults. Furthermore, GBS is one of the leading causes of neonatal and maternal morbidity and mortality. As there is almost complete identity in GBS serotypes isolated from mothers and their infected newborns, it appears that GBS is transmitted from the maternal genital tract to the neonate as it passes through the birth canal. This is a significant source of pathogenic infection for newborns; it is estimated that between 5 % and 30% of pregnant women are infected with GBS. The mortality rate in neonates affected with GBS is 20-25%, with 30% of the infected neonates developing meningitis. Baker, N. Engl. J. Med. 314, 1702 (1986). In addition, GBS accounts for approximately 20% of cases of post-partum endometriosis. Id.
Conventional methods of identifying GBS include gram staining, hemolysis, serological classification on the basis of the Group B antigens, hippurate hydrolysis, the CAMP test, and reaction to bacitracin and sulfamethoxazole-trimethoprim disks. Daly et al., J. Clin. Microbiology 29, 80 (1991). These methods all suffer from low-sensitivity, variable accuracy, and a long turn-around time. Rapid identification of GBS is desirable, especially at the time of parturition, so that both mother and infant can receive prophylactic treatment, thereby preventing the onset of maternal or neonatal disease.
Nucleic acid based diagnostic assays, such as Southern hybridization, offer rapid means of identifying microorganisms, usually in less than one day. Polymerase chain reaction (PCR)-based methods are even more sensitive and can sometimes provide results within hours. However, nucleic acid based methodologies are often fraught with drawbacks. Most of these methods are costly, are available for only a few species of microorganisms, and can resolve only one species per sample tested. Moreover, nucleic acid based assays require the development of oligonucleotide probes or primers that are specific for the microorganism of interest.
To obviate the problems attendant to conventional diagnosis of GBS, there have been attempts to develop nucleic acid based diagnostic methods of identifying GBS.
Baseggio et al., Mol. Cell Probes 11, 349 (1997), provides a method for classifying strains of GBS isolated from mastitic dairy cattle. Genomic DNA isolated from GBS isolates from infected animals is subjected to restriction digestion followed by pulsed-field gel electrophoresis. The electrophoretic profiles of the restriction digests are characteristic for different strains of GBS and provide the basis for diagnosis.
Daly et al., J. Clin. Microbiology 29, 80 (1991), discloses the use of a chemiluminescent-labeled oligonucleotide probe (ACCUPROBE.TM.; Gen-Probe, Inc.) to identify GBS. The DNA probe is targeted to a region of the GBS rRNA. The assay involves hybridizing the probe to target RNA by in-solution hybridization, and then measuring the chemiluminescence. This assay suffers from a low-sensitivity because the target nucleic acids are not amplified prior to hybridization. The infectious microorganisms must be cultured to produce isolated colonies to obtain adequate target rRNA for analysis.
Chatellier et al., J. Clin. Microbiology 35, 2573 (1997), concerns a method for identifying GBS strains in cerebrospinal fluid samples by a randomly-amplified polymorphic DNA (RAPD) assay. Target DNA is amplified by PCR using single primers of random oligonucleotide sequence. The PCR amplification products are separated by electrophoreses, and the resulting gel banding patterns are characteristic of particular GBS strains and are used to make a diagnosis.
U.S. Pat. No. 5,620,847 to Greisen et al. also teaches a PCR-based method for identifying GBS in cerebrospinal fluid. In the first step, a universal bacterial primer is used to amplify a target region in the 16S rRNA. The amplified nucleic acids are then analyzed with a panel of probes, one of which distinguishes GBS from other bacterial species found in cerebrospinal fluid.
Notwithstanding the investigations described above, there remains a need in the art for rapid, accurate and sensitive methods for the identification of GBS.